PS120 Cells Research Articles

Investigation of Calcineurin B Homologous Protein (CHP) Isoform Specific Function

Mammalian cells ubiquitously express the Sodium‐Hydrogen Exchanger Isoform 1 (NHE1) a membrane transporter responsible for intracellular pHi, motility and proliferation. Calcineurin B Homologous Protein (CHP) in part regulates activation and kinetics of the NHE1. CHP has two isoforms, CHP1 and CHP2, whose physiological function remains unclear but both have a unique role in nascent tumor survival. CHP binds NHE via the N and C terminal domains. There is a considerable tertiary structure between both the N terminal domains (amino acids 1–90) and the C terminal domain (aa 111/110–195/196) with a 72% and 74% conserved homology, an intervening loop domain is unique to each CUR. This signature domain CHP unique region (CUR: CHP1 R91 to S110 and CHP2 R91 to S111) when bound to NHE has a specific and distinct structure indicating its potential role in effecting CHP‐NHE1 related functions. The purpose of this study is to investigate how the these CHP unique regions contribute to CHP isoform cell function, specifically cell proliferation and location. Using site directed mutagenesis, we have generated epitope tagged (DDK and MYC) wild type versions of both isoforms of CHP and a variant with the respective CUR domain deleted (CHP1 ΔCUR1 and CHP2ΔCUR2). To distinguish the importance of the CUR domains we have swapped CUR domains between each CHP (CHP2(CUR1) and CHP1(CUR2). These mutations will be expressed in lung fibroblasts expressing NHE1 (CCL39) and PS120 cells (null NHE1 expressing CCL39 derivatives) to determine the impact of each CUR domain on cellular location in the absence and presence of NHE1 and on cell proliferation. We hypothesize that the CUR domain is critical for CHP isoform specific function and NHE1 interactions and present the role of both CHP isoforms on NHE1 related behavior.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

R125H, W240S, C386R, and V507I SLC4A11 mutations associated with corneal endothelial dystrophy affect the transporter function but not trafficking in PS120 cells

SLC4A11 mutations are associated with Fuchs’ endothelial corneal dystrophy (FECD), congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome (endothelial dystrophy with auditory deficiency). Mice with genetically ablated Slc4a11 recapitulate CHED, exhibiting significant corneal edema and altered endothelial morphology. We recently demonstrated that SLC4A11 functions as an NH3 sensitive, electrogenic H+ transporter. Here, we investigated the properties of five clinically relevant SLC4A11 mutants: R125H, W240S, C386R, V507I and N693A, relative to wild type, expressed in a PS120 fibroblast cell line. The effect of these mutations on the NH4Cl-dependent transporter activity was investigated by intracellular pH and electrophysiology measurements. Relative to plasma membrane expression of NaK ATPase, there were no significant differences in plasma membrane SLC4A11 expression among each mutant and wild type. All mutants revealed a marked decrease in acidification in response to NH4Cl when compared to wild type, indicating a decreased H+ permeability in mutants. All mutants exhibited significantly reduced H+ currents at negative holding potentials as compared to wild type. Uniquely, the C386R and W240S mutants exhibited a different inward current profile upon NH4Cl challenges, suggesting an altered transport mode. Thus, our data suggest that these SLC4A11 mutants, rather than having impaired protein trafficking, show altered H+ flux properties.

Proline‐Rich Tyrosine Kinase Phosphorylation's Effect On the Na+/H+ Exchanger Isoform 1

The Na+‐H+ exchanger isoform 1 (NHE1) is a 12‐pass transmembrane protein that functions by exchanging one extracellular sodium ion for one intracellular proton. The transmembrane domain of NHE1 is involved in ion exchange, and the cytoplasmic tail is the regulatory domain which includes a series of phosphorylation sites as well as lipid and proteins binding sites that alter transport activity. NHE1 is a central driver of the hallmarks of cancer through its involvement in the regulation of cellular proliferation, migration, and invasion. While the exact number of phosphorylation sites on the cytoplasmic domain of NHE1 is still unclear, many studies have implicated 22 phosphorylation sites and 12 different kinases. One of the kinases believed to directly or indirectly lead to the phosphorylation of NHE1 at S602 and S605 is proline rich tyrosine kinase 2 (PyK2). To evaluate the role of PyK2 in NHE1 regulation we prepared three cell lines derived from Chinese hamster lung fibroblast lacking NHE1 expression (PS120 cells). These cell lines are PSN (PS120 cells expressing human NHE1), PSN S602A/S605A (PSN cells with serine to alanine mutations of the reported PyK2 phosphorylation sites) and PSN S602D/S605D (PSN cells with serine to aspartic acid mutations of the reported PyK2 phosphorylation sites to mimic phosphorylation). Some studies have found that PyK2 works inversely on NHE1 and causes inhibition upon phosphorylation, while other studies have shown PyK2 to enhance NHE1 activity. Here we evaluate the impact of PyK2 inhibition and mutation of the PyK2 phosphorylation sites on proliferation and migration. Western blot analysis is used to evaluate changes in Pyk2 expression and phosphorylation over time under low and normal serum conditions. Cell proliferation and cell migration are both evaluated. Our results suggest that serum stimulation increases Pyk2 phosphorylation and enhances both cell proliferation and cell migration. Inhibition of PyK2 by PF43I396 or the mutation of the Pyk2 phosphorylation sites on NHE1 decreases proliferation and migration rates in serum stimulated cells.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Effect of SLC4A11 Mutations on Intracellular pH

SLC4A11 is a ubiquitously expressed transporter showing Na+‐OH−, NH3‐H+ cotransport, and water permeability. SLC4A11 mutations have been associated with Fuchs' endothelial corneal dystrophy (FECD), congenital hereditary endothelial dystrophy (CHED2) and Harboyan syndrome (endothelial dystrophy with auditory deficiency). To address the role of known human SLC4A11 mutations on cellular physiology we measured intracellular pH regulation in PS120 fibroblasts that have been stably transfected with SLC4A11 mutant variants.Mutations were made in the Wild‐Type (WT) construct phCMV2‐hSLC4A11 by site‐directed mutagenesis. Mutants with a single amino acid change were obtained and referred to as R125H, C386R, V507I and N639A respectively. PS120 cells were stably transfected with those constructs and empty vector (EV) using SuperFect, and selected by Geneticin. For intracellular pH (pHi) measurement, PS120 cells grown on coverslips were loaded with a pH‐sensitive fluorescent dye, 2′7′‐Bis‐(2‐Carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein (BCECF‐AM). The coverslip was mounted in a perfusion chamber (37°C) on the stage of a fluorescence microscope. Perfusion was performed with a Bicarbonate‐free solution (BF) to obtain stable baseline pH, followed by pulsing cells with 10 mM NH4Cl for 5 minutes, and changed back to BF perfusion until pHi was stable. Fluorescence ratio (495/440 nm) was converted to pHi following calibration with high K+/Nigericin. Results are means ± SE from 3 trials.The baseline pHi was 7.211 ± 0.136, 7.877 ± 0.056, 7.391 ± 0.069, 7.226 ± 0.232, 7.504 ± 0.104 and 7.359 ± 0.221 for WT, EV, R125H, C386R, V507I and N639A, respectively. The pHi increase from NH4Cl pulse was 0.486 ± 0.014, 0.161 ± 0.018, 0.298 ± 0.05, 0.307 ± 0.07, 0.304 ± 0.011 and 0.442 ± 0.084 respectively. The rate of pHi increase (ΔpH/min) was 2.184 ± 0.33, 0.469 ± 0.037, 1.004 ± 0.124, 1.024 ± 0.362, 1.17 ± 0.142 and 1.818 ± 0.405, respectively. The subsequent rate of pHi decrease in the presence of NH4Cl was (ΔpH/min) 0.153 ± 0.038, 0.006 ± 0.012, 0.012 ± 0.005, 0.011 ± 0.003, 0.033 ± 0.002 and 0.009 ± 0.007, respectively. Removal of NH4Cl‐BF resulted in maximum acidification of 1.205 ± 0.122, 0.343 ± 0.074, 0.671 ± 0.019, 0.409 ± 0.122, 0.747 ± 0.041 and 0.748 ± 0.048 pH units. NH4Cl induced pHi changes were WT > N639A > V507I > R125H > C386R > EV.Expression of SLC4A11 WT and mutants lower resting pHi relative to EV, consistent with a H+ permeability. NH4Cl induced pHi changes were consistent with reduction of NH3:H+ linked cotransport in SLC4A11 mutants.Support or Funding InformationNIH R01 EY08834‐23 & P30EY019008

Investigation of the Interaction and Role of CHP1 and CHP2 with NHE1 in Lung Fibroblasts

Intracellular pH (pHi) homeostasis is crucial for cell survival. The membrane protein Sodium‐Hydrogen exchanger 1 (NHE1) is one of the major contributors to a stable pHi. Additionally, NHE1 serves as an anchor for intracellular proteins. NHE1 also plays an essential role in Non‐small cell lung cancer (NSCLC) metastasis as a side effect of the transporter's increased activity to compensate the Warburg effect. Several phosphorylation sites and protein cofactors affect NHE1's activity. The two isoforms calcineurin homologous proteins CHP1 and CHP2 each bind to the C‐terminus of NHE1 and regulate its function. CHP1 is ubiquitous and is key for basel level NHE1 activity while CHP2 is primarily expressed in gut cells, detected in most cancer cells and increases proton transport. To better understand the role of NHE1 and CHP1/CHP2 we generated RFP‐CHP1 and GFP‐CHP2 and transfected each into lung fibroblasts expressing tagged NHE1 (PSN cells) and into lung fibroblasts lacking NHE1 (PS120 cells). Cellular distribution of each CHP was determined and showed significant translocation of CHP1 and CHP2 to the plasma membrane in PSN cells while no localization was observed in PS120 cells. The specific amino acid required for CHP‐NHE1 interaction is determined in cells expressing one of eight different NHE1 amino acid substitutions at the putative CHP‐NHE binding domain of NHE1. To investigate the role of CHP2 binding to NHE we expressed GFP‐CHP2 in PSN and PS120 cells and conducted wound‐healing assays. In addition to cell migration rates, we tracked the migration path of individual cells to assess the impact of each NHE1 mutation on CHP2‐influenced motility.

An Investigation of the Impact of NHE1 Phosphorylation on Steady State Intracellular pH

The Sodium‐Hydrogen Exchanger 1 (NHE1) is involved in cell scaffolding, mobility and intracellular pH (pHi) homeostasis in most cells. The C‐terminus of NHE1 is phosphorylated by several protein kinases, each contributing to NHE1 function. While investigation of one or two sites have been conducted with various agonists, we do not have an understanding of how these sites coordinate or if there is a hierarchical phosphorylation regulation of NHE1. To determine the potential phosphorylation‐dependent regulation of NHE1 on pHi, we focus on four protein kinases with known sites on NHE1; ERK 1⁄2, protein kinase B (AKT), RhoA kinase (Rock) and ribosomal s6 kinase (RSK). To measure the impact of these four kinases we will treat cells with three different agonist; lysophophatidic acid (LPA), platelet‐derived growth factor (PDGF), and phenylephrine (PE). We first determined the dose response for each agonist in fibroblasts deficient in NHE1 expression (PS120 cells) and in stable expressing human NHE1 in PS120 cells (PSN cells). To determine the role of each kinase on NHE1 basal activity, cells were treated with each agonist and teady state pHi was determined 15‐20 min after addition of agonist. Increases in pHi of 0.18 to 0.2 were observed for agonists. The role of each kinase was determined by treating cells prior to agonist with a specific cell permeable kinase inhibitor (1uM MK‐2206, 10uM BI‐D1870, 10uM Y27632 and 0.5uM SCH772984). The impact of direct phosphorylation was determined for each agonist in cells expressing human NHE1 with Ser/Thr to Ala mutations for each kinase site. This work begins to identify the coordinated and role of multiple phosphorylation events on NHE1.

194 N-Glycosylation Is Essential for Ileal ASBT Function and Protection Against Proteases

those patients. Total and surface expression of NHE3 were reduced in rat ileum treated with rapamycin, along with suppressed S6 protein phophorylation and increased ratio of LCII/I, confirming inhibition of mTOR and subsequent activation of autophagy by rapamycin. We further demonstrated that genetic inactivation of autophagy in mouse intestinal epithelium resulted in a marked accumulation of NHE3 and attenuated the rapamycin-induced down-regulation of NHE3, demonstrating a critical role for autophagy in maintaining NHE3 homeostasis. However, in addition to causing autophagic down-regulation of NHE3, we also demonstrate that rapamycin has a second mode of action: at high doses which mimics the sharp rise in serum rapamycin in non-infectious diarrhea, it causes acute inhibition of NHE3 surface expression in mouse ileum and NHE3 transporter activity in fibroblast PS120 cells, lacking endogenous NHE3, when stably transfected with human NHE3. Together, our data implicate two pathophysiological mechanisms of NHE3 down-regulation in the profound non-infectious diarrhea induced by rapamycin: 1) increased autophagic turnover of NHE3 by chronic exposure to rapamycin, and 2) reduced surface expression of NHE3 by acute exposure to a high concentration (spike) of rapamycin. Our findings not only lead to new insights into NHE3 regulation, but also provide new strategies to manage diarrhea in organ transplantations.

Characterization of SLC4A11 as a novel ammonia: 2H+ transporter (893.29)

SLC4A11 mutations cause corneal endothelial dystrophies. This degenerative condition leads to corneal haze, edema, and vision loss. SLC4A11 is a recently identified membrane transporter, of which multiple substrates have been suggested. Intracellular pH measurements in SLC4A11‐expressing PS120 cells revealed that SLC4A11 is NH3/NH4+ and H+ permeable. To further characterize SLC4A11 substrates and transport stoichiometry, we performed patch‐clamp recordings in SLC4A11‐PS120 cells. We found that extracellular 10mM NH3/NH4+ induced inward current, which was inhibited by bath acidification and potentiated by bath alkalinization. Current density (pA/pF) at pHo 6.5, 7.5 and 8.5 were ‐0.22±0.19, ‐6.18±1.09, ‐18.43±3.43, respectively (mean±SE, p<0.001). Reversal potential shift in response to [NH3]o and pHo was consistent with an NH3/H+ coupled transport model. 10mM methylammonium induced smaller current (p<0.05) with similar pH sensitivity. Removal of K+, Na+, Cl‐ did not affect SLC4A11 NH3/H+ transport (K+ p=0.90, Na+ p=0.06, Cl‐ p=0.65), suggesting neither Na+, K+, nor Cl‐ is substrate. Stoichiometry of SLC4A11 NH3:H+ transport was estimated by measuring Erev at different [NH3]o. A plot of Erev against log[NH3]o was linear, and the slope was consistent with a 1:2 stoichiometry. We here identify SLC4A11 as a new member of ammonia channels/transporters, such as AQP8, Rh, MEP, and Amt.Grant Funding Source: Supported by 5R01EY008834(JAB); 5R01HL115140(AGO).

The Cardioprotective Effects of Novel Na+/H+ Exchanger Inhibitor TY-51924 on Ischemia/Reperfusion Injury

The inhibitory effects of sodium 3-guanidinocarbonyl-2-methyl-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-9-ylmethyl sulfate monoethanolate (TY-51924) are selective for Na(+)/H(+) exchanger (NHE)-1 in PS120 cells expressing human NHE isoforms assayed by NH(4)Cl prepulse technique. The median inhibitory concentrations (micromolar) of TY-51924 were 0.095 ± 0.008 (NHE-1), 0.621 ± 0.093 (NHE-2), and >100 (NHE-3). In anesthetized dogs subjected to 90 minutes ischemia/300 minutes reperfusion, intravenous bolus TY-51924 at 5 and 10 mg/kg administered 5 minutes before reperfusion reduced infarct size. The infarct size reduction ratios of TY-51924 at 5 and 10 mg/kg versus vehicle were 32.8% and 52.4%, respectively. But TY-51924 at 10 mg/kg administered 10 minutes after reperfusion did not reduce infarct size. In 2-step intravenous infusion initiated 15 minutes before reperfusion, TY-51924 at low dose (3.8 mg/kg per 5 minutes, then 6.2 mg/kg per 20 minutes) and at high dose (7.6 mg/kg per 5 minutes, then 12.4 mg/kg per 20 minutes) reduced infarct size. The infarct size reduction ratios of TY-51924 at 10 and 20 mg/kg versus vehicle were 39.2% and 51.7%, respectively; plasma drug concentrations at reperfusion were 16.8 and 38.8 μg/mL, respectively. This indicates that maintaining a plasma drug concentration of >20 μg/mL at reperfusion enables TY-51924 to reduce infarct size by inhibiting the NHE, which is activated during the early period of reperfusion.

SLC4A11 is an EIPA-sensitive Na+ permeable pHi regulator

Slc4a11, a member of the solute linked cotransporter 4 family that is comprised predominantly of bicarbonate transporters, was described as an electrogenic 2Na(+)-B(OH)4(-) (borate) cotransporter and a Na(+)-2OH(-) cotransporter. The goal of the current study was to confirm and/or clarify the function of SLC4A11. In HEK293 cells transfected with SLC4A11 we tested if SLC4A11 is a: 1) Na(+)-HCO3(-) cotransporter, 2) Na(+)-OH(-)(H(+)) transporter, and/or 3) Na(+)-B(OH)4(-) cotransporter. CO2/HCO3(-) perfusion yielded no significant differences in rate or extent of pHi changes or Na(+) flux in SLC4A11-transfected compared with control cells. Similarly, in CO2/HCO3(-), acidification on removal of Na(+) and alkalinization on Na(+) add back were not significantly different between control and transfected indicating that SLC4A11 does not have Na(+)-HCO3(-) cotransport activity. In the absence of CO2/HCO3(-), SLC4A11-transfected cells showed higher resting intracelllular Na(+) concentration ([Na(+)]i; 25 vs. 17 mM), increased NH4(+)-induced acidification and increased acid recovery rate (160%) after an NH4 pulse. Na(+) efflux and influx were faster (80%) following Na(+) removal and add back, respectively, indicative of Na(+)-OH(-)(H(+)) transport by SLC4A11. The increased alkalinization recovery was confirmed in NHE-deficient PS120 cells demonstrating that SLC4A11 is a bonafide Na(+)-OH(-)(H(+)) transporter and not an activator of NHEs. SLC4A11-mediated H(+) efflux is inhibited by 5-(N-ethyl-N-isopropyl) amiloride (EIPA; EC50: 0.1 μM). The presence of 10 mM borate did not alter dpHi/dt or ΔpH during a Na(+)-free pulse in SLC4A11-transfected cells. In summary our results show that SLC4A11 is not a bicarbonate or borate-linked transporter but has significant EIPA-sensitive Na(+)-OH(-)(H(+)) and NH4(+) permeability.

Cross‐reactivity of acid‐sensing ion channel and Na+–H+ exchanger antagonists with nicotinic acetylcholine receptors

Nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout the mammalian central and peripheral nervous systems, where they contribute to neuronal excitability and synaptic communication. It has been reported that nAChRs are modulated by BK channels and that BK channels, in turn, are inhibited by acid-sensing ion channels (ASICs). Here we investigate the possible functional interaction between these channels in medial habenula (MHb) neurones. We report that selective antagonists of large-conductance calcium-activated potassium channels and ASIC1a channels, paxilline and psalmotoxin 1, respectively, did not induce detectable changes in nicotine-evoked currents. In contrast, the non-selective ASIC and Na(+)-H(+) exchanger (NHE1) antagonists, amiloride and its analogues, suppressed nicotine-evoked responses in MHb neurones of wild-type and ASIC2 null mice, excluding a possible involvement of ASIC2 in the nAChR inhibition by amiloride. Zoniporide, a more selective inhibitor of NHE1, reversibly inhibited α3β4-, α7- and α4-containing (*) nAChRs in Xenopus oocytes and in brain slices, as well as in PS120 cells deficient in NHE1 and virally transduced with nAChRs, suggesting a generalized effect of zoniporide in most neuronal nAChR subtypes. Independently from nAChR antagonism, zoniporide profoundly blocked synaptic transmission onto MHb neurones without affecting glutamatergic and GABA receptors. Taken together, these results indicate that amiloride and zoniporide, which are clinically used to treat hypertension and cardiovascular disease, have an inhibitory effect on neuronal nAChRs when used experimentally at high doses. The possible cross-reactivity of these compounds with nAChRs in vivo will require further investigation.

Serum- and glucocorticoid-induced kinase 3 in recycling endosomes mediates acute activation of Na+/H+exchanger NHE3 by glucocorticoids

Na(+)/H(+) exchanger 3 (NHE3) is the major Na(+) transporter in the intestine. Serum- and glucocorticoid-induced kinase (SGK) 1 interacts with NHE regulatory factor 2 (NHERF2) and mediates activation of NHE3 by dexamethasone (Dex) in cultured epithelial cells. In this study, we compared short-term regulation of NHE3 by Dex in SGK1-null and NHERF2-null mice. In comparison to wild-type mice, loss of SGK1 or NHERF2 significantly attenuated regulation of NHE3 by Dex but did not completely obliterate the effect. We show that transfection of SGK2 or SGK3 in PS120 cells resulted in robust activation of NHE3 by Dex. However, unlike SGK1 or SGK2, SGK3 rapidly activated NHE3 within 15 min of Dex treatment in both PS120 and Caco-2bbe cells. Immunofluorescence analysis showed that SGK3 colocalized with NHE3 in recycling endosomes, whereas SGK1 and SGK2 were diffusely distributed. Mutation of Arg-90 of SGK3 disrupted the endosomal localization of SGK3 and delayed NHE3 activation. Activation of SGK3 and NHE3 by Dex was dependent on phosphoinositide 3-kinase (PI3K) and phosphoinositide-dependent kinase 1 (PDK1), and Dex induced translocation of PDK1 to endosomes. Our study identifies SGK3 as a novel endosomal kinase that acutely regulates NHE3 in a PI3K-dependent mechanism.

The Differential Role of SGK3 from SGK1 and SGK2 in Activation of NHE3 by Glucocorticoid

NHE3 is the major Na+/H+ transporter located in the apical membrane of epithelial cells in the intestine and renal proximal tubule. We have previously shown that SGK1 is involved in activation of NHE3 by dexamethasone (DEX). In this study, we examined whether SGK2 and SGK3 are also involved in DEX stimulation of NHE3 as both SGK2 and SGK3 are expressed in the small intestine. We used PS120/NHE3V and Caco-2BBe/hNHE3V cells as the models. Overexpression of either SGK2 or SGK3 in PS120/NHE3V cells caused an increase in NHE3 activity by potentiating exocytosis of NHE3 to the plasma membrane. Importantly, in contrast to SGK1 and SGK2, SGK3-activated NHE3 occurred as early as 15 min in both PS120 and Caco2-BBe cells after DEX treatment. Similarly, kinase assay showed that SGK3 but not SGK1 or SGK2 activity was clearly increased at 15 min after DEX. Immunofluorescence staining showed that SGK3 is located in endosomes, whereas SGK1 and SGK2 are ubiquitously distributed throughout the cell. Interestingly, a mutation of SGK3 (R90A) caused disruption of its endosomal localization and delays of NHE3 activation. In addition, we showed that activation of SGK3 and SGK3-mediated NHE3 activation by DEX are dependent on glucocorticoid receptor (GR) and PI3K. In conclusion, the endosomal location of SGK3 makes it distinct from SGK1 and SGK2 by mediating an early response of NHE3 to DEX in a GR-PI3K dependent manner.

Impact of NHE1 Phosphorylation by Ribosomal S‐6 kinase and RhoA kinase on ERM binding and cell motility

Cellular migration involves complex signaling and intricate reorganization of the cytoskeleton. Ezrin/Radixin/Moesin (ERM) proteins play a crucial role in the reorganization of the cytoskeleton by binding to membrane bound proteins including the Na+‐H+ exchanger (NHE) and anchoring stress fibers to the plasma membrane. During cellular migration, there is a reorganization of the membrane to localize NHE to the leading edge of the cell. Localization of NHE helps to give the cell polarity, which is important in directing stress fiber formation to assist the cell to move forward. The focus of this study was to determine the conditions by which ERM translocates to the membrane to bind to NHE and examine the role of the ERM:NHE binding complex in proper formation of unidirectional cell migration. We have prepared a series of Ser or Thr – Ala mutants at putative RhoA Kinase (Rock) and Ribosomal S‐6 kinase (RSK) phosphorylation sites and expressed these in NHE1‐null PS120 cells. Co‐immunoprecipitation of NHE1 identified ERM‐NHE1 interactions for each mutant following activation with lysophopsphatidic acid (LPA). Using wild‐type and non‐phosphorylatable EFGP‐ERM, we investigated the localization of ERM proteins and the impact of NHE1 phosphorylation. Finally we show the effect of NHE1 and ERM phosphorylation on stress fiber formation and cell motility.. This work was supported with funds from NSF‐MCB‐081778, NSF‐RUI‐MCB 0930432, and NIH‐1‐R15‐CA135616‐01.

Nuclear accumulation of Calcineurin B Homologous Protein 2 (CHP2) results in enhanced proliferation of tumor cells

The interaction between calcineurin B homologous protein 2 (CHP2) and Na(+) /H(+) exchanger 1 (NHE1), two membrane proteins, is essential for protecting cells from serum deprivation-induced death. Although four putative EF-hands in CHP2 had been predicted for years, Ca²(+) -binding activities of these motifs have not been tested yet, their role in this process remain poorly understood. To identify Ca²(+) -binding motifs required for the stable formation of CHP2/NHE1 complexes, we developed a mutagenesis-based assay in PS120 cells. We found that (45) Ca²(+) bond to two EF-hand motifs (EF3 and 4) of CHP2 proteins with high affinity. Complex formation between CHP2 and the CHP2 binding domain of NHE1 resulted in a marked increase in the Ca²(+) -binding affinity of CHP2. Co-immunoprecipitation and distribution of GFP-tagged CHP2-EF3m/4m also indicated that Ca²(+) affected the membrane location of CHP2 to interact with NHE1. The C-terminal region of CHP2 contains a nuclear export sequence (NES). When the six leucines of NES were mutated to alanines, the resulting CHP2 protein was predominantly localized to the nucleus. Furthermore, mutation of the NES resulted in enhanced proliferation and oncogenic potential of HeLa cells. Together, these results show that calcium and NES control the subcellular distribution of CHP2 and then distinctively regulate cell proliferation.

NHE3 Activity Is Dependent on Direct Phosphoinositide Binding at the N Terminus of Its Intracellular Cytosolic Region

The small intestinal BB Na(+)/H(+) antiporter NHE3 accounts for the majority of intestinal sodium and water absorption. It is highly regulated with both postprandial inhibition and stimulation sequentially occurring. Phosphatidylinositide 4,5-bisphosphate (PI(4,5)P(2)) and phosphatidylinositide 3,4,5-trisphosphate (PI(3,4,5)P(3)) binding is involved with regulation of multiple transporters. We tested the hypothesis that phosphoinositides bind NHE3 under basal conditions and are necessary for its acute regulation. His(6) proteins were made from the NHE3 C-terminal region divided into four parts as follows: F1 (amino acids 475-589), F2 (amino acids 590-667), F3 (amino acids 668-747), and F4 (amino acids 748-832) and purified by a nickel column. Mutations were made in the F1 region of NHE3 and cloned in pet30a and pcDNA3.1 vectors. PI(4,5)P(2) and PI(3,4,5)P(3) bound only to the NHE3 F1 fusion protein (amino acids 475-589) on liposomal pulldown assays. Mutations were made in the putative lipid binding region of the F1 domain and studied for alterations in lipid binding and Na(+)/H(+) exchange as follows: Y501A/R503A/K505A; F509A/R511A/R512A; R511L/R512L; R520/FR527F; and R551L/R552L. Our results indicate the following. 1) The F1 domain of the NHE3 C terminus has phosphoinositide binding regions. 2) Mutations of these regions alter PI(4,5)P(2) and PI(3,4,5)P(3) binding and basal NHE3 activity. 3) The magnitude of serum stimulation of NHE3 correlates with PI(4,5)P(2) and PI(3,4,5)P(3) binding of NHE3. 4) Wortmannin inhibition of PI3K did not correlate with PI(4,5)P(2) or PI(3,4,5)P(3) binding of NHE3. Two functionally distinct phosphoinositide binding regions (Tyr(501)-Arg(512) and Arg(520)-Arg(552)) are present in the NHE3 F1 domain; both regions are important for serum stimulation, but they display differences in phosphoinositide binding, and the latter but not the former alters NHE3 surface expression.

βPix Up-regulates Na+/H+ Exchanger 3 through a Shank2-mediated Protein-Protein Interaction

Na(+)/H(+) exchanger 3 (NHE3) plays an important role in neutral Na(+) transport in mammalian epithelial cells. The Rho family of small GTPases and the PDZ (PSD-95/discs large/ZO-1) domain-based adaptor Shank2 are known to regulate the membrane expression and activity of NHE3. In this study we examined the role of betaPix, a guanine nucleotide exchange factor for the Rho GTPase and a strong binding partner to Shank2, in NHE3 regulation using integrated molecular and physiological approaches. Immunoprecipitation and pulldown assays revealed that NHE3, Shank2, and betaPix form a macromolecular complex when expressed heterologously in mammalian cells as well as endogenously in rat colon, kidney, and pancreas. In addition, these proteins co-segregated at the apical surface of rat colonic epithelial cells, as detected by immunofluorescence staining. When expressed in PS120/NHE3 cells, betaPix increased membrane expression and basal activity of NHE3. Interestingly, the effects of betaPix on NHE3 were abolished by cotransfection with dominant-negative Shank2 mutants and by treatment with Clostridium difficile toxin B, a Rho GTPase inhibitor, indicating that Shank2 and Rho GTPases are involved in betaPix-mediated NHE3 regulation. Knockdown of endogenous betaPix by RNA interference decreased Shank2-induced increase of NHE3 membrane expression in HEK 293T cells. These results indicate that betaPix up-regulates NHE3 membrane expression and activity by Shank2-mediated protein-protein interaction and by activating Rho GTPases in the apical regions of epithelial cells.

Regulation of Na+/H+ Exchanger Isoform 1 (NHE1) by Calmodulin-binding Sites: Role of Angiotensin II

We examined the effect of Angiotensin II (Ang II) on the interaction between the Ca²⁺/CaM complex and hNHE1. Considering that calmodulin binds to NHE1 at two sites (A and B), amino acids at both sites were modified and two mutants were constructed: SA^1K3R/4E and SB^1K3R/4E. Wild type and mutants were transfected into PS120 cells and their activity was examined by H⁺ flux (J_H+). The basal J_H+ of wild type was 4.71 ± 0.57 (mM/min), and it was similar in both mutants. However, the mutations partially impaired the binding of CaM to hNHE1. Ang II (10^-12 and 10^-9 M) increased the J_H+ in wild type and SB. Ang II (10^-6 M) increased this parameter only in SA. Ang II (10^-9 M) maintained the expression of calmodulin in wild type or mutants, and Ang II (10^-6 M) decreased it in wild type or SA, but not in SB. Dimethyl-Bapta-AM (10^-7 M), a calcium chelator, suppressed the effect of Ang II (10^-9 M) in wild type. With Ang II (10^-6 M), Bapta failed to affect wild type or SA, but it increased the J_H+ in SB. W13 or calmidazolium chloride (10^-5 M), two distinct calmodulin inhibitors, decreased the effect of Ang II (10^-9 M) in wild type or SB. With Ang II (10^-6 M), W13 or calmidazolium chloride decreased the J_H+ in wild type or SA and increased it in SB. Thus, with Ang II (10^-12 and 10^-9 M), site A seems to be responsible for the stimulation of hNHE1 and with Ang II (10^-6 M), site B is important to maintain its basal activity.

NHERF3 (PDZK1) Contributes to Basal and Calcium Inhibition of NHE3 Activity in Caco-2BBe Cells

Elevated intracellular Ca(2+) ([Ca(2+)](i)) inhibition of NHE3 is reconstituted by NHERF2, but not NHERF1, by a mechanism involving the formation of multiprotein signaling complexes. To further evaluate the specificity of the NHERF family in calcium regulation of NHE3 activity, the current study determined whether NHERF3 reconstitutes elevated [Ca(2+)](i) regulation of NHE3. In vitro, NHERF3 bound the NHE3 C terminus between amino acids 588 and 667. In vivo, NHE3 and NHERF3 associate under basal conditions as indicated by co-immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer. Treatment of PS120/NHE3/NHERF3 cells, but not PS120/NHE3 cells, with the Ca(2+) ionophore, 4-bromo-A23187 (0.5 mum): 1) inhibited NHE3 V(max) activity; 2) decreased NHE3 surface amount; 3) dissociated NHE3 and NHERF3 at the plasma membrane by confocal immunofluorescence and fluorescence resonance energy transfer. Similarly, in Caco-2BBe cells, NHERF3 and NHE3 colocalized in the BB under basal conditions but after elevation of [Ca(2+)](i) by carbachol, this overlap was abolished. NHERF3 short hairpin RNA knockdown (>50%) in Caco-2BBe cells significantly reduced basal NHE3 activity by decreasing BB NHE3 amount. Also, carbachol-mediated inhibition of NHE3 activity was abolished in Caco-2BBe cells in which NHERF3 protein expression was significantly reduced. In summary: 1) NHERF3 colocalizes and directly binds NHE3 at the plasma membrane under basal conditions; 2) NHERF3 reconstitutes [Ca(2+)](i) inhibition of NHE3 activity and dissociates from NHE3 in fibroblasts and polarized intestinal epithelial cells with elevated [Ca(2+)](i); 3) NHERF3 short hairpin RNA significantly reduced NHE3 basal activity and brush border expression in Caco-2BBe cells. These results demonstrate that NHERF3 reconstitutes calcium inhibition of NHE3 activity by anchoring NHE3 basally and releasing it with elevated Ca(2+).

IRBIT, Inositol 1,4,5-Triphosphate (IP3) Receptor-binding Protein Released with IP3, Binds Na+/H+ Exchanger NHE3 and Activates NHE3 Activity in Response to Calcium

Calcium (Ca2+) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca2+ regulates the activity of Na+/H+ exchanger 3 (NHE3). However, the effect of Ca2+-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP3 receptor-binding protein released with IP3 (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca2+-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca2+-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca2+-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca2+ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition.